基質金屬蛋白酶-7水解凝血調節素之衍生片段參與表皮細胞株之表皮-間葉過渡研究

Abstract

凝血脢調節素(TM)最初發現於血管壁內皮細胞表面的一種醣蛋白，初期此蛋白質具有天然內皮層抗凝血素之特性。不過，近年來的研究著重於凝血脢調節素的表現量與上皮細胞的上皮-間質轉化(EMT)兩者的關聯性。為了研究基質金屬蛋白酵素7(MMP-7)在上皮細胞進行上皮-間質轉化時，凝血脢調節素受蛋白質水解時可能扮演的角色，本實驗建立了一個共同轉染有凝血脢調節素與基質金屬蛋白酵素7兩者表現質體的細胞株(MDCK)。利用西方墨點分析法確認了凝血脢調節素與基質金屬蛋白酵素7的表現量後，觀察到一個有趣的結果，即在共轉染有凝血脢調節素與基質金屬蛋白酵素7兩基因的MDCK細胞中，凝血脢調節素會受到金屬水解酵素所切割。另外，從核質分離試驗中，本實驗發現了凝血脢調節素片段中的一個分子量約為34 kDa的蛋白質片段，的確存在於細胞核內，從共軛焦顯微鏡之影像分析結果，也確認了此凝血脢調節素片段確實會分布於細胞核。 在胚胎發育過程中會發生上皮-間質轉化現象，而與其相關之腫瘤形成之初，也具有此轉換之現象。在上皮-間質轉化的過程之中，SNAIL蛋白的表現量與腫瘤細胞的移動性與侵犯性之強弱有很高的關聯性。從初步的實驗結果看來，在MDCK細胞株中，基質金屬蛋白脢7所切割後而形成之人類凝血調節素片段，會與SNAIL蛋白進入細胞核機制有關，進而調控下游各種基因的表達，包括凝血調節素本身基因。然而，SNAIL蛋白與細胞質中的凝血脢調節素片段如何形成複合體，進而轉移到細胞核中和其背後所代表的生物功能仍需被釐清。

Thrombomodulin (TM) is a glycoprotein that was originally identified in vascular endothelium and characterized as a natural endothelial anticoagulant [1]. From recent studies[2] [3] [4], it is focusing on the role of TM during Epithelial-mesenchymal transition (EMT). In this study, we investigated the potential role of MMP-7 in proteolysis of TM protein during EMT. An epithelial cell line, MDCK, was transfected with TM and/or MMP-7. Using western blotting analysis the expression of MMP7 proteins could make TM being proteolytically processed with formation of multiple fragments of TM in the TM and MMP7-cotransfected MDCK. Furthermore, from the nuclear v.s. cytoplasmic fraction assay, we demonstrated that the major ~34 kDa fragment was seen in the nuclear fraction. Confocal microscopy image analysis also further confirmed the nuclear localization of TM fragments. Epithelial-mesenchymal transition (EMT) occurs during embryonic development and may also be responsible for onset of EMT-associated tumorigenesis. During EMT, SNAIL protein is up-regulated and its expression levels are strongly correlated with increased motility and invasion of cells [5] [6]. From the preliminary results, we found that the processing of human thrombomodulin protein by MMP-7 in MDCK cells, which may be associated with translocation of SNAIL protein into nucleus to regulate its target genes [7], including TM gene activation. However, the biological functions of nuclear-translocation of the ternary complex of SNAIL and cytosol TM are still unclear and need to be further elucidated in the future.